Changes in the microflora composition of goat and sheep milk during lactation

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INTRODUCTION

Milk due to its beneficial composition and properties is

suitable environment for the development of

contaminating microorganisms. A relatively low number of microorganisms is contained in the milk of healthy animals mainly tending to settle in the teat canal and passes into the milk tank of udder. The number of microorganisms in milk ranges from 101 to 103 respectively 104 CFU x mL-1 in the process of the leaving udder. A 105 CFU x mL-1 could be obtained in the milk from the animal with the textured flaccid sphincter. The bacteria of the genus Micrococcus, Enterococcus, coryneform bacteria are represented, and infrequently e.g. staphylococci. In the diseased animals, e.g. Streptococcus agalactiae, S. dysgalactiae, Staphylococcus aureus, E. coli, and Klebsiella etc. can also occur (Hejlová, 1997;

Görner and Valík, 2004). During milking, the milk is exposed to secondary contamination from the environment of the milking equipment, hands of the milker, and udder etc. (Hejlová, 1997; Malá et al., 2010). The quality of milk hygiene is microbiologically significant, which varies in terms of the types and numbers according to climate,

weather, grazing, lactation, housing, health, herd management etc. According to Švejcarová et al. (2010), inferior microbiological parameters may prove a negative effect on the final product. When the bacteria content is too low, not only pathogenic microflora can be disturbed but also the natural non-pathogenic one, which can significantly affect the properties of dairy products made from raw milk (Kalantzopoulos, 2003).

The criteria for hygienic quality of raw milk are listed in the Regulation of the European Parliament and Council Regulation (EC) no. 853/2004 as amended. The microorganism content at 30 °C (per mL) should be ≤1500000 in goat and sheep milk (rolling geometric mean over a two-month period, at least two samples per month). However, if the milk is intended for the production of dairy products from raw milk by a process without heat treatment, the milk should contain ≤500 000 microorganisms per mL. These regulations are the only applicable microbiological criteria in the Czech Republic for raw goat and sheep milk.

doi:10.5219/489

CHANGES IN THE MICROFLORA COMPOSITION OF GOAT

AND SHEEP MILK DURING LACTATION

Libor Kalhotka, Lenka Dostálová, Květoslava Šustová, Jan Kuchtík, Lenka Detvanová

ABSTRACT

The aim of this work was to determine extend of microbial contamination of raw milk in individual seasons. Raw goat milk (3 farms) and sheep milk (2 farms) were analyzed. Milk was produced on farms of different way of farming and with a different number of milked animals. Samples were taken during lactation three terms in the beginning, middle and end of lactation. In milk, following groups of microorganisms were determined by standard methods: total count of microorganisms (TCM), psychrotrophic microorganisms, Enterobacteriaceae, lactic acid bacteria (lactobacilli), enterococci, aerobic and anaerobic thermoresistant microorganisms (TMRae, TMRan), micromycetes (yeast and moulds). In goat milk, the following numbers of microorganisms were detected: total count of microorganisms (TCM) from 105 to 109 CFU x mL-1, lactobacilli from 102 to 105 CFU x mL-1, bacteria fam. Enterobacteriaceae from 101 to 105 CFU x mL-1, enterococci from 101 to 105 CFU x mL-1, thermoresistant aerobic and anaerobic microorganisms (TMRae and TMRan) from units to 103 resp. 105 CFU x mL-1, psychrotrophic microorganisms from 101 to 106 CFU x mL-1, micromycets from 101 to 104 CFU x mL-1. In the sheep milk, the following numbers of microorganisms were determined: TCM from 105 to 106 CFU x mL-1, lactobacilli from 103 to 106 CFU x mL-1, bacteria fam. Enterobacteriaceae from 101 to 105 CFU x mL-1, enterococci from 101 to 104 CFU x mL-1, TMRae and TMRan from units to 105 CFU x mL-1, psychrotrophic microorganisms from 104 to 106 CFU x mL-1, micromycets from 102 to 104 CFU x mL-1. From the above mentioned results, the following conclusions can be suggested. The bacterial counts of raw goat and sheep milk are highly variable and influenced by a number of important factors in the course of lactation and year (temperature, health, secondary contamination etc.). The bacterial numbers are not affected by the stage of lactation. High numbers of microorganisms in goat and sheep milk may be primarily caused by the insufficient cleaning and sanitizing of milking equipment or low hygiene of hand milking. An important role may also act the cooling rate of milk and purity of cooling eguipment.

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The aim of this study was to find out the dynamics of the microflora composition of raw goat and sheep milk from different farm breeds during lactation.

MATERIAL AND METHODOLOGY

Raw goat milk (3 farms) and sheep milk (2 farms) were analyzed. Milk was produced on farms of different way of farming and with a different number of milked animals (Tab. 1 and 2). Samples were taken during lactation in the beginning (spring), middle (summer) and end of lactation (autumn). In milk, following groups of microorganisms were determined by standard methods: total count of

microorganisms (TCM) on PCA-AB medium

(MILCOM – Tábor, Czech Rep.) at 30 °C for 72 h,

psychrotrophic microorganisms on PCA medium

(MILCOM – Tábor, Czech Rep.) at 6.5 °C for 10 days,

fam. Enterobacteriaceae on VRBG medium

(MILCOM – Tábor, Czech Rep.) at 37 °C for 24 h, lactobacilli on MRS medium (MILCOM – Tábor, Czech Rep.) anaerobic cultivation at 37 °C for 48 h, enterococci on Slanetz-Bartley Agar (Merck, Germany) at 37 °C 48 h, aerobic and anaerobic thermoresistant microorganisms (TMRae, TMRan) after thermoinactivation (85 °C 10 min.) on PCA at 37 °C for 48 h, micromycetes (yeast and moulds) on YGC medium (MILCOM - Tábor, Czech Rep.) at 25 °C for 120 h. After the cultivation of particular Petri dishes accrued colonies were counted and the result was expressed in CFU x mL-1.

RESULTS AND DISCUSSION

The results of microbiological analyzes of goat and sheep milk samples are shown in Table 3. In goat milk, the

following numbers of microorganisms were detected: total count of microorganisms (TCM) from 105 to 109 CFU x mL-1, lactobacilli from 102 to 105 CFU x mL-1, bacteria fam. Enterobacteriaceae from 101 to 105 CFU x mL-1, enterococci from 101 to 105 CFU/mL, thermoresistant aerobic and anaerobic microorganisms (TMRae and TMRan) from units to 103 resp. 105 CFU x mL-1, psychrotrophic microorganisms from 101 to 106 CFU x mL-1, micromycets from 101 to 104 CFU x mL-1. In the sheep's milk, the following numbers of microorganisms were determined: TCM from 105 to 106 CFU x mL-1, lactobacilli from 103 to 106 CFU x mL-1, bacteria fam.

Enterobacteriaceae from 101 to 105 CFU x mL-1, enterococci from 101 to 104 CFU x mL-1, and TMRae and TMRan from units to 105 CFU x mL-1, psychrotrophic microorganisms from 104 to 106 CFU x mL-1 micromycets from 102 to 104 CFU x mL-1.

TCM is only one legislative covered group of microorganisms in milk of small ruminants. The numbers are regulated by the European Parliament and Council Regulation (EC) no. 853/2004 and the maximum of ≤1.5 million cells should be contained in 1 mL. The average values of two-year monitoring for individual farms and lactation period are shown in Fig. 1. The results show that the limit value was multiply exceeded in the monitoring period for most of the farms. The exception is the farm no. 1, for which the limit value is not exceeded in any single case and TCM values ranged from 104 to 105 CFU x mL-1. This state of farm no. 1 is held in the long term (Kalhotka et al., 2013). The observed values of TCM of farm no. 1 prove to be very close to the average values of 1.1 x 105 CFU x mL-1 as indicated Kouřimská

Table 1 Chacterization of farms.

Farm Typ of milk Breed Type of farming Milking

animals Region

1 goat White Shorthaired Goat conventional 131 dist. Blansko (SMR)

2 goat White Shorthaired Goat conventional 18 dist. Prostějov (OR)

3 goat Crossbreed of Anglo-Nubian goat organic 20 dist. Vyškov (SMR)

4 sheep Lacaune conventional 83 dist. Šumperk (OR)

5 sheep Lacaune organic 100 dist. Vsetín (ZR)

dist. – district, SMR – South Moravian Region, OR – Olomouc Region, ZR – Zlín Region

Table 2 Selected parameters relating to milking on farms.

Farm Milking per day

Toilet m.

gland Parlor

Milking

post-treatment Cooling equipment

1 2x wet parallel no cooling equipment

2 2x wet parallel barrier spec. glasses in the refrigerator

3 2x wet spec. box no spec. glasses in the refrigerator

4 2x wet parallel barrier cooling equipment

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and Dvořáková (2008). TCM fluctuated during lactation between the farms and it can not be clearly said that TCM rose with the increasing duration of lactation. At the same time, significant annual differences were also found out up to two logarithmic orders. Nevertheless, the individual

milk samples obtained from healthy animals contained small numbers of microorganisms, as for instance mentioned Králíčková et al. (2011), TCM can achieve high numbers in mixed samples. The average values of TCM for the whole period are shown in Fig. 2. Even here,

Table 3 Counts of microorganisms in goat and sheep milk in CFU x mL-1.

Farm Seasons Year TCM Lbc. Entero. Ent. TMRae TMRan Psych. Mikromyc.

Farm spring 2013 2.8 × 105 3.7 × 103 2.0 × 103 3.5 × 102 11 1 2.0 × 104 2.1 × 103 1 2014 2.1 × 105 4.6 × 103 7.1 × 103 22 28 23 1.5 × 105 1.9 × 103 summer 2013 2.3 × 105 1.2 × 104 1.6 × 103 4.4 × 102 23 28 8.4 × 104 2.9 × 103 2014 9.5 × 105 3.2 × 105 1.6 × 104 1.7 × 102 24 21 2.6 × 105 2.5 × 103 autumn 2013 7.0 × 104 8.6 × 103 30 2.5 × 102 9.8 × 103 4.2 × 103 3.7 × 103 9.1 × 102 2014 7.7 × 105 3.9 × 102 2.7 × 104 6.6 × 102 2 1 2.2 × 105 1.6 × 103 Farm spring 2013 2.3 × 107 9.5 × 105 5.6 × 103 1.2 × 105 2.9 × 103 9.8 × 102 4.7 × 106 1.9 × 104 2 2014 9.5 × 105 2.4 × 103 3.2 × 102 8.7 × 102 1.4 × 105 20 2.3 × 105 1.2 × 103 summer 2013 1.1 × 105 2.7 × 102 11 2.0 × 102 8 1 3.5 × 103 1.6 × 102 2014 1.7 × 106 1.0 × 105 3.8 × 104 1.7 × 104 23 16 4.3 × 105 6.9 × 103 autumn 2013 1.1 × 107 8.4 × 105 7.2 × 104 1.6 × 105 7.8 × 103 82 1.1 × 106 3.2 × 103 2014 3.0 × 106 1.4 × 103 6.9 × 103 7.7 × 102 5.6 × 102 3.8 × 102 2.1 × 105 1.0 × 104 Farm spring 2013 7.1 × 105 6.0 × 103 3.6 × 103 61 5 0 1.2 × 106 1.6 × 103 3 2014 1.4 × 105 5.3 × 103 3.9 × 102 94 20 2.2 × 102 5.1 × 103 65 summer 2013 2.3 × 105 3.0 × 102 2.6 × 102 32 3 1 <100 23 2014 5.2 × 106 5.4 × 104 9.2 × 105 3.9 × 103 1.1 × 102 90 1.4 × 104 1.5 × 103 autumn 2013 1.2 × 109 2.1 × 103 7.9 × 102 5.4 × 102 10 15 7.0 × 103 2.7 × 103 2014 3.7 × 107 1.3 × 104 1.6 × 104 6.0 × 104 3.3 × 102 92 1.5 × 106 7.8 × 102 Farm spring 2013 3.8 × 106 9.2 × 104 4.5 × 104 3.8 × 102 58 1.2 × 102 1.3 × 106 1.7 × 104 4 2014 5.3 × 106 1.1 × 105 6.6 × 103 1.6 × 103 5.1 × 103 3.6 × 103 1.6 × 106 5.7 × 103 summer 2013 3.8 × 105 1.4 × 105 4.7 × 103 2.2 × 103 15 9 1.6 × 104 1.1 × 104 2014 8.2 × 105 1.3 × 105 4.6 × 104 1.8 × 104 32 20 6.7 × 105 3.5 × 104 autumn 2013 5.9 × 106 2.2 × 103 2.1 × 105 5.4 × 102 1.1 × 105 1.5 × 105 1.2 × 106 5.0 × 104 2014 2.5 × 106 6.5 × 103 6.8 × 103 1.4 × 104 90 7.1 × 102 6.6 × 105 4.8 × 104 Farm spring 2013 4.5 × 105 1.8 × 103 4.0 × 103 95 48 5 1.3 × 104 1.6 × 102 5 2014 3.7 × 106 1.9 × 104 <100 9.5 × 103 8.0 × 102 13 3.5 × 104 3.4 × 102 summer 2013 1.1 × 106 1.8 × 104 1.4 × 103 2.2 × 104 8 11 4.5 × 104 4.9 × 102 2014 4.0 × 106 2.3 × 106 3.8 × 104 9.9 × 104 2.1 × 103 57 8.1 × 105 1.7 × 103 autumn 2013 5.2 × 106 1.1 × 106 2.3 × 103 1.1 × 104 5.3 × 102 4.0 × 102 2.2 × 106 2.5 × 104 2014 4.5 × 106 1.8 × 103 5,5 × 102 6.1 × 102 3.7 × 102 2.7 × 102 2.5 × 105 1.5 × 103 TCM –Total count of microorganisms, Lbc. – lactobacilli, Entero. – fam. Enterobacteriaceae, Ent. – enterococci, TMRae – aerobic thermoresistant microorganisms, TMRan – anaerobic thermoresistant microorganisms, Psychro. m. – psychrothrophic microorganisms, mikromyc. – micromycetes (yeasts and moulds)

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it can be easy to see that the best results were achieved at the farm no. 1.

In goat milk, the highest average values of TCM were detected at the farm no. 3 using an environmentally friendly method. For sheep's milk, no significant difference was found out between the farms using

conventional or organic method.

The recommended values based on CSN 570529 related to cow's milk can be used for the evaluation of other

monitored microorganism groups also proving

technological importance. The number of psychrotrophic microorganisms should be up to 50000 in 1 mL,

Figure 1 The average value of TCM in each seasons (2013 - 2014) for farms 1-5.

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heat-resistant microorganisms up to 2000 in 1 mL, the highest number of coliforms up to 1000 in 1 mL, spore anaerobic bacteria should be negative in 0.1 mL. These microbiological limits are not determined for milk of small ruminants. In goat and sheep milk, the numbers are expected proportionately higher. Our identified microbial counts are corresponding with the findings (Tab. 3). High numbers TCM and coliform bacteria also mentioned

Oliveira et al. (2011) and Morgan et al. (2003).

The numbers of enterococci, lactobacilli but also coliform bacteria, yeasts and TCM samples of raw goat milk are lower up to several orders which are presented in the study by Foschino et al. (2002), where significant differences are indicated between the farms.

High numbers of microorganisms in milk can cause spoilage unless the milk is quickly and efficiently cooled and subsequently pasterized before further processing. In pasteurized milk, where the majority of contaminating microflora is destroyed, present microbial enzymes remaining active can cause the spoilage except to surviving microorganisms. The surviving microorganisms or microbial enzymes are also active and can spoil dairy products, which are made from such milk.

The results of microbiological analyzes show that the number of microorganisms in milk has been extensively individual and changeable. The final microbiological quality of the milk is caused by a number of factors

(Hejlová, 1997; Kalantzopoulos, 2003; Morgan et al., 2003; Malá et al., 2010; Cempírková et al., 2012). The stage of lactation (Tab. 3, Fig. 1) did not prove a significant effect on the number of microorganisms in goat and sheep milk opposite to the number of somatic cells corresponding to the data of Foschino et al. (2002) and

Švejcarová et al. (2010). High numbers of microorganisms in goat and sheep milk may be primarily caused by the insufficient cleaning and sanitizing of milking equipment or low hygiene of hand milking. An important role may also act the cooling rate of milk and purity of cooling eguipment. The method for cooling milk (cooling equipment versus the use of special glasses, see Tab. 2) may play an important role, especially in the farms from 1 to 3 producing goat milk.

CONCLUSION

From the above mentioned results, the following conclusions can be suggested. The bacterial counts of raw goat and sheep milk are highly variable and influenced by a number of important factors in the course of lactation and year. The bacterial numbers are not affected by the stage of lactation. Due to the small sample size and short term of monitoring, it can not be clearly said which farming method is preferable in the terms of microbial milk contamination. More important role acts the access, care and responsibility of the farmer apart from the mentioned factors. Not only for this reason, the microbiological analysis should be repeated for the following periods.

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Acknowledgment:

This work was supported by grant NAZV KUS QJ1230044.

Contact address:

Libor Kalhotka, Mendel University in Brno, Faculty of Agronomy, Department of Agrochemistry, Soil Science, Microbiology and Plant Nutrition, Zemědělská 1, 613 00

Brno, Czech Republic, E-mail:

xkalhotk@node.mendelu.cz

Lenka Dostálová, Mendel University in Brno, Faculty of Agronomy, Department of Agrochemistry, Soil Science, Microbiology and Plant Nutrition, Zemědělská 1, 613 00

lenka.dostalova@mendelu.cz

Květoslava Šustová, Mendel University in Brno, Faculty of Agronomy, Department of Food Technology, Zemědělská 1, 613 00 Brno, Czech Republic, E-mail: kvetoslava.sustova@mendelu.cz

Jan Kuchtík, Mendel University in Brno, Faculty of Agronomy, Department of Animal Breeding, Zemědělská

1, 613 00 Brno, Czech Republic, E-mail:

jan.kuchtik@mendelu.cz

Lenka Detvanová, Mendel University in Brno, Faculty of Agronomy, Department of Agrochemistry, Soil Science, Microbiology and Plant Nutrition, Zemědělská 1, 613 00